Targeting and modification of prokaryotic cell–cell junctions by tobacco mosaic virus cell‐to‐cell movement protein

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.     

Genetic evidence of an essential role for cytomegalovirus small capsid protein in viral growth

Many steps in the replication cycle of cytomegalovirus (CMV), like cell entry, capsid assembly, and egress of newly synthesized virions, have not been completely analyzed yet. In order to facilitate these studies, we decided to construct a recombinant CMV that incorporates the green fluorescent protein (GFP) into the nucleocapsid. A comparable herpes simplex virus type 1 (HSV-1) mutant has recently been generated by fusion of the GFP open reading frame (ORF) with the HSV-1 ORF encoding small capsid protein (SCP) VP26 (P. Desai and S. Person, J. Virol. 72:7563-7568, 1998). Recombinant CMV genomes expressing a fusion protein consisting of GFP and the SCP were constructed by the recently established bacterial artificial chromosome mutagenesis procedure. In transfected cells, the SCP-GFP fusion protein localized to distinct foci in the nucleus that may represent sites for capsid assembly (assemblons). However, no viable progeny was reconstituted from these mutant CMV genomes. CMV genomes with deletion of the SCP ORF also did not give rise to infectious virus. Rescue of the mutation by insertion of the SCP gene at an ectopic position in an SCP knockout genome indicates that, in contrast to the HSV-1 SCP, the CMV SCP is essential for viral growth. Expression of the SCP-GFP fusion protein together with the authentic SCP blocked the CMV infection cycle, suggesting that the SCP-GFP fusion protein exerts a dominant-negative effect on the assembly of new virions. The results of this study are discussed with regard to recently published data about the structure of the CMV virion and its differences from the HSV-1 virion.     

Genetic evidence for an essential role of brassinosteroids in plant development

Brassinosteroids which show high structural similarity to animal steroid hormones elicit a variety of growth responses when exogeneously applied to plant tissues. Thus far however, the function of endogeneous brassinosteroids in higher plants has been unclear. This paper describes three extremely dwarfed Arabidopsis thaliana mutants, cbb1 (dwf1-6), cbb2 and cbb3, which are impared in cell elongation controlled by brassinosteroids. While cbb1 (dwf1-6) and cbb3 can be phenotypically normalized to wild-type by feeding with brassinosteroids indicating deficiencies of brassinosteroid biosynthesis, cbb2 is brassinosteroid-insensitive and defines a function required for further metabolic conversion necessary for biological activity or for perception/signal transduction of these growth-regulating plant steroid hormones. Expression of the meri5 and TCH4 genes is low in all three cbb mutants and can be restored to wild-type levels by brassinosteroid treatment in the cbb1 (dwf1-6) and cbb3 mutants but are unaffected in the cbb2 mutant. These data indicate that brassinosteroids are essential for proper plant development and play an important role in the control of cell elongation.     

Genetic depletion reveals an essential role for an SR protein splicing factor in vertebrate cells

SR proteins are essential for the splicing of messenger RNA precursors in vitro, where they also alter splice site selection in a concentration-dependent manner. Although experiments involving overexpression or dominant mutations have confirmed that these proteins can influence RNA processing decisions in vivo, similar results with loss-of-function mutations have been lacking. Now, a system for genetic depletion of the chicken B cell line DT40 has revealed that the SR protein ASF/SF2 (alternative splicing factor/splicing factor 2) is essential for viability in these cells⁽¹⁾. This study opens the way for a complete functional dissection of this protein, and other SR proteins, in vivo.     

Xylem‐to‐phloem transfer of boron in broccoli and lupin during early reproductive growth

The aim of this study was to test the hypothesis that newly‐acquired boron (B) undergoes rapid xylem‐to‐phloem transfer in plants with restricted mobility. Analysis of the element accumulation and water usage by shoots of intact broccoli ( Brassica oleracea var. italica Plenck cv. Commander) and lupin ( Lupinus albus L. cv. Ultra) plants provided with a non‐deficient supply of B, revealed that the concentration of various mineral elements (K, P, Mg, Ca, B, Fe, Zn, Mo, Cu, Mn) in xylem sap of intact plants ranged from 0.3 µ M to 3.5 m M , with B being present at 2.9‐3.5 µ M . For each element assayed, the concentration was higher in phloem exudate (1.6 µ M to 91 m M ) than in xylem sap; B was present at about 0.4 m M . Intact broccoli and lupin plants or detached transpiring broccoli shoots were supplied simultaneously with enriched ¹⁰B, strontium (a xylem marker) and rubidium (a xylem/phloem marker) during early reproductive growth. The contents of these three compounds were determined in foliage and florets or fruits as a function of time (i.e. up to 12 h and 4 days for broccoli and lupin plants, respectively), and the content in florets or fruits was expressed as a percent of the total recovered. In general, the percent recovery of both ¹⁰B and rubidium in florets or fruits was similar and markedly greater than that for strontium, even at the earliest harvest times (within 2 h for broccoli and 1 day for lupin). The data indicate that in plants with restricted B mobility, B is supplied to sink tissues in the phloem, and the extent of B xylem‐to‐phloem transfer is closely determined by current uptake.     

Genetic evidence strongly support an essential role for PfPV1 in intra-erythrocytic growth of P. falciparum

Upon invading the host erythrocyte, the human malaria parasite P. falciparum lives and replicates within a membrane bound compartment referred to as the parasitophorous vacuole. Recently, interest in this compartment and its protein content has grown, due to the important roles these play in parasite egress and protein traffic to the host cell. Surprisingly, the function of many proteins within this compartment has not been experimentally addressed. Here, we study the importance of one of these proteins, termed PfPV1, for intra-erythrocytic parasite survival. Despite numerous attempts to inactivate the gene encoding PfPV1, we were unable to recover deletion mutants. Control experiments verified that the pv1 gene locus was per se open for gene targeting experiments, allowing us to exclude technical limitations in our experimental strategy. Our data provide strong genetic evidence that PfPV1 is essential for survival of blood stage P. falciparum, and further highlight the importance of parasitophorous vacuole proteins in this part of the parasite's life cycle.     

Diffusion in the littoral zone: scoping emergence times and movement to essential habitat for young‐of‐year brook trout in lakes

Factors governing the process of dispersal of lake‐spawned brook trout ( Salvelinus fontinalis ) young‐of‐year (YOY) appear drastically different from those governing dispersal in more commonly studied stream salmonids. Rather than dispersal being highly density‐dependent and the result of territoriality and aggression, in brook trout it may be density‐independent and driven by a common need for coldwater habitat for summer survival. Emerging fish travel great distances from single spawning sites on lakes to cold groundwater habitat. Movement is a gradual, one‐dimensional diffusion around lake margins and represents a very unique and simplified natal dispersal pathway allowing for dispersal rates, distances, and factors controlling these parameters to be measured. We predicted timing of the emergence period for brook trout alevins from lake spawning sites using the known relation between water temperature and emergence times for salmonids. Emergence and dispersal was then observed in the field by visually estimating YOY density in segments of the littoral zone throughout the dispersal period. Fluxes in density in these sections were used to estimate rate of YOY spread. YOY behaviour and body condition was also assessed across the wave of spatial spread. We are working towards a spatially explicit model to identify critical groundwater rearing habitat needing protection from forestry activities.     

Nickel in higher plants: further evidence for an essential role

Soybeans (Glycine max [L.] Merr.) grown in Ni-deficient nutrient solutions accumulated toxic urea concentrations which resulted in necrosis of their leaflet tips, a characteristic of Ni deficiency. Estimates of the Ni requirement of a plant were made by using seeds produced with different initial Ni contents. When compared to soybeans grown from seeds containing 2.5 nanograms Ni, plants grown from seeds containing 13 nanograms Ni had a significantly reduced incidence of leaflet tip necrosis. Plants grown from seeds containing 160 nanograms Ni produced leaves with almost no leaflet tip necrosis symptoms. Neither Al, Cd, Sn, nor V were able to substitute for Ni.

In other experiments, a small excess of EDTA was included in the nutrient solution in addition to that needed to chelate micronutrient metals. Under these conditions, nodulated nitrogen-fixing soybeans had a high incidence of leaflet tip necrosis, even when 1 micromolar NiEDTA was supplied. However, in nutrient solutions containing inorganic sources of N, 1 micromolar NiEDTA almost completely prevented leaflet tip necrosis, although no significant increase in leaf urease activity was observed. Cowpeas (Vigna unguiculata [L.] Walp) grown in Ni-deficient nutrient solutions containing NO₃ and NH₄ also developed leaflet tip necrosis, which was analogous to that produced in soybeans, and 1 micromolar NiEDTA additions prevented these symptoms.

These findings further support our contention that Ni is an essential element for higher plants.

     

A new method to evaluate egg‐to‐fry survival in salmonids, trials with Atlantic salmon

A new method to estimate the survival of salmonids from egg fertilization to fry emergence is described. Fine mesh screen cylindrical capsules, 12 cm³ in volume, filled with batches of 10 eggs of Atlantic salmon Salmo salar were implanted in the substratum using small removable guiding tubes inserted with a metal spike. The method was compared with two other commonly used techniques, capping redds with fry‐traps and fine mesh screen incubation‐emergence boxes buried into the gravel. Egg‐to‐fry survival was recorded for the three methods run in parallel in artificial redds created on three sites of the Nivelle River watershed (south‐west of France), two in the stream and one in an experimental spawning channel. In the channel, survival to the eyed stage and to hatching in capsules and incubation‐emergence boxes was also compared. The implant of capsules proved easier and faster to use than other methods, the structure of the surrounding substratum was less disturbed and the capsules were less vulnerable to spates. This technique provided survival values largely free of the bias induced by other methods and intermediate between that of incubators and of traps.     

The cytoplasmic-localized, cytoskeletal-associated RNA binding protein OsTudor-SN: evidence for an essential role in storage protein RNA transport and localization

Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of Os Tudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. Os Tudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that Os Tudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that Os Tudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of Os Tudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of Os Tudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged Os Tudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of Os Tudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that Os Tudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.     

Genetic evidence for the essential role of beta-transducin repeat-containing protein in the inducible processing of NF-kappa B2/p100

Processing of the nf kappa b2 gene product p100 to generate p52 is an important step in NF-kappa B regulation. This step is regulated by a nonclassical NF-kappa B signaling pathway involving the NF-kappa B-inducing kinase (NIK). NIK induces p100 processing by triggering phosphorylation of specific C-terminal serines of p100. However, the downstream molecular events leading to p100 processing remain unclear. Here we show that NIK induced the physical recruitment of beta-transducin repeat-containing protein (beta-TrCP), a component of the SCF ubiquitin ligase complex, to p100. This event required the phosphorylation sites as well as the death domain of p100. Using the RNA interference technique, we demonstrated that beta-TrCP is essential for NIK-induced p100 ubiquitination and processing. Interestingly the constitutive processing of p100 mutants was independent of beta-TrCP. These results suggest that beta-TrCP is an essential component of NIK-induced p100 processing.     

Genetic evidence for an evolutionarily conserved role of IL-7 signaling in T cell development of zebrafish

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens.     

Genetic evidence for an inhibitory role of tomosyn in insulin‐stimulated GLUT4 exocytosis

Exocytosis is a vesicle fusion process driven by soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs). A classic exocytic pathway is insulin‐stimulated translocation of the glucose transporter type 4 (GLUT4) from intracellular vesicles to the plasma membrane in adipocytes and skeletal muscles. The GLUT4 exocytic pathway plays a central role in maintaining blood glucose homeostasis and is compromised in insulin resistance and type 2 diabetes. A candidate regulator of GLUT4 exocytosis is tomosyn, a soluble protein expressed in adipocytes. Tomosyn directly binds to GLUT4 exocytic SNAREs in vitro but its role in GLUT4 exocytosis was unknown. In this work, we used CRISPR‐Cas9 genome editing to delete the two tomosyn‐encoding genes in adipocytes. We observed that both basal and insulin‐stimulated GLUT4 exocytosis was markedly elevated in the double knockout (DKO) cells. By contrast, adipocyte differentiation and insulin signaling remained intact in the DKO adipocytes. In a reconstituted liposome fusion assay, tomosyn inhibited all the SNARE complexes underlying GLUT4 exocytosis. The inhibitory activity of tomosyn was relieved by NSF and α‐SNAP, which act in concert to remove tomosyn from GLUT4 exocytic SNAREs. Together, these studies revealed an inhibitory role for tomosyn in insulin‐stimulated GLUT4 exocytosis in adipocytes. We suggest that tomosyn‐arrested SNAREs represent a reservoir of fusion capacity that could be harnessed to treat patients with insulin resistance and type 2 diabetes.     

Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during potyvirus infection

The Arabidopsis thaliana-potyvirus system was developed to identify compatibility and incompatibility factors involved during infection and disease caused by positive-strand RNA viruses. Several Arabidopsis mutants with increased susceptibility to Tobacco etch potyvirus (TEV) were isolated previously, revealing a virus-specific resistance system in the phloem. In this study, Arabidopsis mutants with decreased susceptibility to Turnip mosaic potyvirus (TuMV) were isolated. Three independent mutants that conferred immunity to TuMV were isolated and assigned to the same complementation group. These mutants were also immune or near-immune to TEV but were susceptible to an unrelated virus. The locus associated with decreased susceptibility was named loss-of-susceptibility to potyviruses 1 (lsp1). The LSP1 locus was isolated by map-based cloning and was identified as the gene encoding translation factor eIF(iso)4E, one of several known Arabidopsis isoforms that has cap binding activity. eIF4E and eIF(iso)4E from different plant species were shown previously to interact with the genome-linked protein (VPg) of TEV and TuMV, respectively. Models to explain the roles of eIF(iso)4E during virus infection are presented.     

Genetic evidence for glucitol-specific enzyme III, an essential phosphocarrier protein of the Salmonella typhimurium glucitol phosphotransferase system.

Positive selection procedures were developed for the isolation of mutants defective in components of the glucitol-specific catabolic enzyme system in Salmonella typhimurium. gutA (enzyme IIgut-negative), gutB (enzyme IIIgut-negative), and gutC (constitutive for the glucitol operon) mutants were isolated and characterized biochemically and genetically. The gene order was shown to be gutCAB.     

Involvement of the cylindrical inclusion (CI) protein in the overcoming of an eIF4E-mediated resistance against Lettuce mosaic potyvirus

The capacity of Lettuce mosaic virus to overcome the lettuce resistance conferred by the mo1¹ and mo1² alleles of the gene for eukaryotic translation initiation factor 4E (eIF4E) was analysed using reverse genetics. Mutations in the virus genome-linked protein (VPg) allowed mo1¹ only to be overcome, but mutations in the C-terminal portion of the cylindrical inclusion (CI) protein allowed both alleles to be overcome. Site-directed mutagenesis pinpointed a key role of the amino acid at position 621 in the virulence. This is the first example of the involvement of a potyviral CI protein in the breaking of an eIF4E-mediated resistance.     

Paclobutrazol inhibition of sterol biosynthesis in a cell suspension culture and evidence of an essential role for 24-ethylsterol in plant cell division

Growth of a celery (Apium gravidens) cell suspension culture was inhibited by the synthetic plant growth regulator paclobutrazol. Paclobutrazol caused a reduction in the incorporation of [2-14C]acetate into the 4-demethyl sterols (campesterol, sitosterol, stigmasterol) but radioactivity accumulated in the 4 alpha-methylsterols. The accumulating 4 alpha-methylsterols were identified as obtusifoliol and cycloeucalenol indicating that paclobutrazol was inhibiting sterol biosynthesis by blocking 14 alpha-demethylation. The inhibition of celery cell growth by paclobutrazol could be partially overcome by addition of cholesterol to the culture medium. However, addition of stigmasterol restored growth to the control value suggesting an essential role for a 24-ethylsterol to support plant cell division.     

Genetic evidence of a coupling role for the TraG protein family in bacterial conjugation

The ability of conjugative plasmids from six different incompatibility groups to mobilize a set of mobilizable plasmids was examined. The mobilization frequencies of plasmids RSF1010, ColE1, ColE3, and CloDF13 varied over seven orders of magnitude, depending on the helper conjugative plasmid used. Mobilization of CloDF13 was unique in that it did not require TrwB, TraG or TraD (all members of the TraG family) for mobilization by R388, RP4 or F, respectively. CloDF13 itself codes for an essential mobilization protein (MobB) which is also a TraG homolog, only requiring a source of the genes for pilus formation. Besides, CloDF13 was mobilized efficiently by all conjugative plasmids, suggesting that TraG homologs are the primary determinants of the mobilization efficiency of a plasmid, interacting differentialy with the various relaxosomes. Previous results indicated that TraG and TrwB were interchangeable for mobilization of RSF1010 and ColE1 by PIL_W (the pilus system of IncW plasmids) but TraG could not complement conjugation of trwB mutants, suggesting that additional interactions were taking place between TrwB and oriT(R388) that were not essential for mobilization. To further test this hypothesis, we analyzed the mobilization frequencies of ColE1 and RSF1010 by the P, W, and F pili in the presence of alternative TraG homologs. The results obtained indicated that the frequency of mobilization was determined both by the particular TraG-like protein used and by the pilus system. Thus, TraG-like proteins are not generally interchangeable for mobilization. Therefore we suggest that the factors that determine the frequencies of transfer of different MOB regions are the differential interactions of TrwB with pilus and relaxosome. Received: 9 September 1996 / Accepted: 17 December 1996     

RNA helicase activity of the plum pox potyvirus CI protein expressed in Escherichia coli. Mapping of an RNA binding domain.

The plum pox potyvirus (PPV) cylindrical inclusion (CI) protein fused to the maltose binding protein (MBP) has been synthesized in Escherichia coli and purified by affinity chromatography in amylose resin. In the absence of any other viral factors, the fusion product had NTPase, RNA binding and RNA helicase activities. These in vitro activities were not affected by removal of the last 103 amino acids of the CI protein. However, other deletions in the C-terminal part of the protein, although leaving intact all the region conserved in RNA helicases, drastically impaired the ability to unwind dsRNA and to hydrolyze NTPs. A mutant protein lacking the last 225 residues retained the competence to interact with RNA. Further deletions mapped boundaries of the RNA binding domain within residues 350 and 402 of the PPV CI protein. This region includes the arginine-rich motif VI, the most carboxy terminal conserved domain of RNA helicases of the superfamily SF2. These results indicate that NTP hydrolysis is not an essential component for RNA binding of the PPV CI protein.